Project Summary/Abstract Alcohol use disorder (AUD) is a pervasive public health problem that carries great personal and economic costs. Despite the serious nature of AUD, we lack a thorough understanding of the brain mechanisms involved in excessive alcohol consumption. Therefore, a significant goal for future research is to more precisely map the molecular and circuit adaptations that accompany escalated alcohol intake. Although there are several models that allow mechanistic investigation of how escalated alcohol drinking can alter brain circuits and signaling, this project will focus on the intermittent access (IA) to alcohol paradigm, as it reliably drives high levels of voluntary alcohol intake and withdrawal during alcohol deprivation. While numerous neurochemical systems have been identified as playing a role in AUD, one of the most promising leads for therapeutic intervention is the kappa opioid receptor (KOR) and its endogenous ligand dynorphin (Dyn). The Dyn/KOR system is upregulated in both alcohol-dependent humans and rodents repeatedly exposed to alcohol. Further, Dyn/KOR signaling has been shown to contribute to escalated alcohol intake and negative-affective states associated with alcohol withdrawal. Consistent with the important role of the Dyn/KOR system in excessive alcohol intake, I have generated preliminary results that show pharmacological blockade of KOR suppresses escalated alcohol drinking in the IA paradigm. One possible brain region underlying this effect is the insular cortex (IC). Though the IC is a highly understudied brain region in the context of alcohol research, several studies have shown the IC is involved in alcohol self-administration and undergoes structural adaptations following high levels of alcohol intake. In my preliminary work, I have identified a discrete subpopulation of Dyn-expressing pyramidal cells in layer 2/3 of the IC (ICDyn) that are engaged by long-term IA to alcohol. In addition, I have found that Dyn decreases excitability in KOR-expressing layer 5 interneurons, and that long-term IA to alcohol increases excitatory drive in IC layer 5 pyramidal cells. This indicates that KOR signaling locally modulates layer 5 neuronal activity and IA to alcohol may induce plasticity within an ICDyn laminar microcircuit. The proposed experiments will thoroughly characterize how long-term IA to alcohol impacts this newly identified ICDyn microcircuit by integrating multiple converging electrophysiological, pharmacological, and optogenetic approaches. Using these approaches, I will map the connectivity of this ICDyn microcircuit and assess adaptations in basal excitability and Dyn/KOR signaling in the IC that accompany long-term IA to alcohol drinking. I will also localize KOR to distinct subpopulations of neurons in layer 5 of the IC using a multiplexed fluorescence in situ hybridization assay. By generating a greater mechanistic understanding of how this novel ICDyn microcircuit is impacted by escalated alcohol intake, I will inform future studies aimed at mitigating alcohol abuse and dependence. Ultimately, this proposal will identify a site-dependent, cell-specific, and signaling- selective mechanism that can be used for the targeted treatment of AUD.